Investigating the detrimental effect of homocysteine and the regulation of transsulfuration pathway in kidney ischemia -reperfusion injury

Ischemia followed by reperfusion is a major cause for renal injury in both the native kidney and renal allograft. Hyperhomocysteinemia is independently associated with allograft loss in kidney transplanted patients. The aim of the first part of the study was to determine the role of homocysteine (Hcy) in ischemia-reperfusion-induced renal injury. The left kidney of a Sprague-Dawley rat was subjected to ischemia alone or ischemia followed by different reperfusion periods. Hcy levels were elevated 2.9- and 1.5-fold in kidneys subjected to ischemia alone or ischemia-reperfusion, respectively. Administration of anti-Hcy antibodies into the kidney not only abolished ischemia-reperfusion-induced oxidative stress and cell death in kidneys but also restored renal function after 1-h of reperfusion. Further investigation revealed that elevation of Hcy level in the kidney upon ischemia-reperfusion was due to reduced activity of cystathionine-β-synthase (CBS), a key enzyme in Hey metabolism. The aim of the second part of the study was to investigate the effects of pH and nitric oxide (NO) on the CBS activity in the kidney during ischemia-reperfusion. The pH was markedly reduced in kidneys upon ischemia due to metabolic acidosis. Injection of alkaline solution into the kidney partially restored the CBS activity. Further analysis revealed that the reduction of CBS activity during reperfusion was accompanied by a significant elevation of nitric oxide synthase activity and NO metabolites (nitrate and nitrite) in the kidney tissue. Injection of a NO scavenger, restored the CBS activity in kidneys subjected to ischemia-reperfusion.;The aim of the third part of the study was to characterize the endogenous production of H2S by the rat kidney. Of total CBS activity, 1/3 was committed in the H2S producing pathway. Both Hcy and cysteine (Cys) were utilized as substrates in this pathway however, Cys was found to be the limiting substrate. Ischemia-reperfusion resulted in a significant reduction of the CBS activity in the H2S producing pathway as well. These results suggested that metabolic acidosis during ischemia and accumulation of NO metabolites during reperfusion contributed to reduced CBS activity in both the standard transsulfuration pathway as well as the H2S producing pathway leading to an elevation of renal Hcy levels, which in turn, played a detrimental role in the kidney.